Malaria is a disease caused by protozoan parasites that are transmitted to humans by mosquitoes in the genus Anopheles. There are nearly 500 million new cases of malaria every year, and from 1-2 million people die from the disease while others are severely debilitated. About half the human population is at risk of contracting malaria, and its range may spread as global warming accelerates. The broad, long term objectives of this proposal are to create new methods of combating malaria to complement the current methods of control, namely insecticides to kill mosquito vectors and drugs to kill parasites in infected people. This project seeks to develop the means to create strains of bacteria that can interfere with the ability of mosquitoes to transmit malaria thus reducing its overall health burden and aiding in the goal of eradicating this disease. The specific aims of this research project are as follows: Aim 1: Creation of strains of Asaia SF2.1 that secrete anti-Plasmodium effector proteins using native secretion signals. Asaia SF2.1 is intimately associated with Anopheles mosquitoes in the field, colonizing the midgut, salivary glands, and gonads of these insects. Thus it has optimal microbial ecology on which to build a paratransgenesis system against malaria. We will develop native secretion systems using genomic data and a genetic screen for the secretion of anti-malarial effector proteins from Asaia SF2.1 to ensure efficient secretion of these proteins in mosquito midguts. Aim 2: Isolation of strong conditional promoters from Asaia SF2.1. Antimalarial effector proteins must be expressed when the parasite is present in the mosquito midgut and in sufficient quantities to be effective. We will isolate strong conditional promoters from Asaia SF2.1 that are active when bacteria are present during a blood meal using a genetic screen, genomic homology searches, and RNAseq. Aim 3: Creation of genetically stable strains of transgenic Asaia SF2.1. For eventual field use, paratransgenic strains of bacteria must be genetically stable. They cannot be based on laboratory plasmids that are maintained with drug selection. We will develop methods to create strains that contain genes inserted in the chromosome or borne on plasmids that need no drug selection.